The range over which leu mRNA and enzyme levels vary in Escherichia coli is much larger than previously suspected (greater than 1000 fold). We propose to investigate the mechanisms underlying this wide range of control by four types of studies. 1. In vivo studies with E. coli K12. These will be directed towards answering the following two questions: a) Is the entire range of expression of the leucine operon under control by leucine or is some other signal (for example, guanosine tetraphosphate) involved? b) Is the leucine operon regulated in part by a mechanism involving mRNA termination? 2. In vitro transcription of the leucine operon of E. coli. Using short, endonuclease-generated fragments of DNA that contain the leu promoter, we will determine whether transcription of the leucine operon requires only RNA polymerase and nucleoside triphosphates or whether some positive factor is required in addition. 3. In vitro transcription and translation of the leucine operon of E. coli in a Zubay system. We have developed sensitive new radioassays and used them to demonstrate in vitro coordinate synthesis of two leu enzymes programmed by linear and circular templates. We will use this system to investigate the reason for increased efficiency of circular molecules as templates and to search for conditions and factors which specifically inhibit and stimulate expression of the leucine operon in vitro. 4. Studies with the leucine operon of Salmonella typhimurium. The leucine operon of S. typhimurium (for which a good genetic map exists) has been cloned on plasmid pMB9. After developing techniques for transferring mutations from the chromosome to the plasmid, we will be able to construct a detailed restriction endonuclease map. This in turn will make possible a determination of nucleotide sequences of interesting regions of the operon.